Genomic and functional impact of Trp53 inactivation in JAK2V617F myeloproliferative neoplasms

Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53−/− mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53−/−) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53−/−-reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.


INTRODUCTION
Classical myeloproliferative neoplasms (MPNs) are hematopoietic disorders characterized by clonal proliferation of mature myeloid elements, which manifest clinically as excess red blood cells, platelets, and/or white blood cells (WBCs) [1].Mutations in kinases such as Janus kinase 2 (JAK2) have been identified in the majority of patients with MPNs, underscoring the importance of activated transduction signaling in the pathogenesis of these disorders [2][3][4][5][6].A role of cellular tumor antigen p53 (p53) in JAK2V617F signaling-induced cell proliferation has been postulated.JAK2 strongly inhibits the stabilization of p53 after induction of DNA damage through the increase in E3 ubiquitinprotein ligase Mdm2 (MDM2) translation, which is dependent on the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway in cell lines and primary cells from MPN patients [7].Moreover, JAK2V617F-dependent cell growth is inhibited by MDM2 inhibitors, thus suggesting an important role of the MDM2/ p53 axis in JAK2V617F-mediated proliferation [8,9].
However, inactivation and mutations in TP53 have also been associated with post-MPN acute myeloid leukemia (AML) transformation (in 25%-50% of cases) [10][11][12][13], suggesting that inactivation of the TP53 tumor suppressor could be a cooperating contributing event in combination with signaling due to JAK2 gain-of-function mutation during the transformation process.
Preclinical data from TP53-inactivated mouse models overexpressing JAK2V617F suggest that the combination of this sole abnormality in a JAK2V617F-overexpressing cell could be sufficient to cause acute transformation in 1.5 to 4 months [14,15], but during the MPN phase development, the MPN phenotype of JAK2V617F/Trp53 −/− mice does not seem to be highly different from the phenotype of JAK2V617F-only mice.This finding was confirmed recently by Li et al. using a transgenic mouse model [16].However, in this model, when JAK2V617F expression is only driven by its promoter, transformation occurs after a latency of 135 days.This latency suggests that p53 inactivation could be more involved in genetic instability and a long-term transformation process than in short-term leukemic transformation.Moreover, clinical data tempered the "2-hits only" hypothesis because AML is uncommon (∼5% of associated malignancies) in patients with Li-Fraumeni syndrome associated with germline TP53 mutations [17,18].Additionally, patients with chronic-phase MPN harboring TP53 mutations who have a long survival time do not have an increased risk of transformation [19][20][21].
Recent studies suggest that the quantity of TP53-mutated cells could be responsible for the transformation, more so than the existence of TP53 mutation; small TP53-mutated clones (less than 10% of Variant allele frequency) could have a neutral impact while larger clones could be detrimental [19][20][21][22].
To better define the role of TP53 in the JAK2V617F phenotype and the impact of TP53 inactivation in MPN evolution and the response to treatment to reduce the clonal size, we studied mice with Trp53 inactivation and JAK2V617F endogenous mutation presenting a full phenotype of MPN (i.e., thrombocytosis, polycythemia, and leukocytosis).
Here, our data suggest that (1) p53 may be un-responsible for the MPN progressive phenotype, despite a large set of gene expression deregulations being affected by Trp53 inactivation; (2) Trp53 inactivation facilitates clonal progression; and (3) Trp53 inactivation induces resistance to interferon-α (IFN-α) therapy in MPN but is not sufficient to induce direct transformation of chronic-phase MPN, leading us to hypothesize that acute transformation of such pathologies requires more than two oncogenic hits.

Mouse model
Mice were bred and maintained in pathogen-free conditions in the animal facilities unit at the Université de Paris Cité.All procedures performed has been carried out in accordance with national (n°2013-118) and European (n°2010/63/UE) directives for animal experiments.Animals were handled according to the guidelines of institutional animal care committees using protocols approved by the "Comité d'Ethique Experimentation Animal Paris-Nord" (no.121) (project number #24499-2020030501008440).Animals were housed at our animal facility (UMS Saint-Louis US53/UAR2030, Institut de Recherche Saint-Louis, Paris, France) in accordance with animal welfare and ethical guidelines (accreditation number B75-10-08).

BM and spleen analysis
Mice were euthanized by cervical dislocation.Bones (femurs and tibias) and spleens were harvested, and muscle and tendon tissue were removed using a scalpel and Kimwipes.BM and spleen samples were used for cell count, cytometry, and histology.
BM fraction was flushed out using a syringe containing 1× PBS with 2% fetal bovine serum (FBS).The resulting cell suspension was filtered through a 40-µm cell strainer (Corning, NY, USA) and pelleted by centrifugation.For mechanical grinding, spleens were smashed and ground between rough sides of frosted glass slides, and cells were collected in DMEM containing 2% FBS.Cell suspensions were passed through a 40-µm cell strainer, and cells were re-suspended in DMEM containing 10% FBS.Suspensions were subjected to cytometry analysis after red blood cell lysis.
Murine pegylated-IFN-α (PharmaEssentia Corp, Taiwan) was subcutaneously injected every week at doses of 600 ng in 0.1 mL phosphate buffered-saline (PBS)/mouse.Alternatively, 100 µL of 10 mg/mL bromodeoxyuridine (BrdU) were intraperitoneally injected (BD Pharmingen, USA).Orbital plexus blood from anesthetized mice was collected in ethylenediaminetetraacetic acid tubes.Blood cell counts were determined using an automated blood counter (MS9, Schloessing Melet, Cergy-Pontoise, France).Blood samples were used for cytometry analysis.BM cells were collected by flushing both femurs and tibias.Spleens were weighed, and single-cell suspensions were prepared.
For proliferation analysis, surface staining was performed as described above, and cells were processed for cell proliferation according to the manufacturer's recommendations (BD Pharmingen BrdU Flow kits) with Alexa Fluor 488 anti-BrdU Antibody, 3D4 (BioLegend).Data analysis was performed at the Cochin Cytometry and Immunobiology Facility Cytometry analyses on a Fortessa LSRII cytometer (BD Biosciences), and analyses were done on Kaluza software.
After the paired end (2 × 75 bp) sequencing, a primary analysis based on AOZAN software (ENS, Paris) was applied to demultiplex and to control the quality of the raw data (based on FastQC modules / version 0.11.5).The fastq files were then aligned using the STAR algorithm (version 2.7.1a) on the GRCm38 reference from Ensembl (release 101) and quality control of the alignment realized with Picard tools (version 2.8.1).STAR parameters were the following: --sjdbOverhang 74 --twopassMode Basic --outFilter-Type BySJout --quantMode TranscriptomeSAM.Reads were then counted using RSEM (v1.3.1) with the AlignedtoTranscriptome bam files, and the statistical analyses on the read counts were performed with the DESeq2 package version 1.24.0 to determine the proportion of differentially expressed genes between 2 conditions.During the statistical analysis, we filtered out annotations where there were less than 3 samples with normalized counts greater than or equal to 10.
Pre-ranked lists of gene fold changes were generated in DESeq2 between 3 groups.Analyses were pursued with fast gene set enrichment analysis (FGSEA) implemented in the R Bioconductor package applied to the ranked gene lists.Principal component analysis (plotPCA), volcano plots (EnhancedVolcano), and gesaplots were all generated in R.

Constitutive expression of JAK2V617F/Vav-Cre in Trp53inactivated cells results in MPN-like phenotype
The JAK2V617F conditional allele mice were crossed with Vav-Cre transgenic mice to generate JAK2V617F/Vav-Cre mice.To assess the function of Trp53 inactivation in the background of JAK2V617F, the JAK2V617F/Vav-Cre/Trp53 −/− mice were bred as described in the mouse model section.
Histological spleen analysis revealed that JAK2V617F/Vav-Cre mice lost their classical histology: removal of white pulp, significant expansion of red pulp, and increased number of megakaryocytes.Increased number and size of clusters of immature red lineage precursors were noticed in the 2 types of mice (data not shown).
No evidence of blast infiltration was noticed at 1 and 3 months old in blood, BM, or spleen samples in both JAK2V617F mice and JAK2V617F/Vav-Cre/Trp53 −/− mice (Fig. S1).
Lastly, survival analysis showed that the median survival time for JAK2V617F/Vav-Cre mice was around 2 months when no treatment (or venesection) was performed; Trp53 inactivation did not shorten JAK2V617F/Vav-Cre mice's (already short) lifespan (Fig. 1F).
Collectively, these data show that heterozygous endogenous JAK2V617F expression in hematopoietic cells leads to hyperplasia of mature and maturing erythroid, granulocytic, and megakaryocytic cells in blood and hematopoietic tissues, an MPN phenotype that may not be influenced by the P53 genetic inactivation.
2 months and half-life expectancy of engrafted mice was reduced to 100 to 120 days (Fig. 3C).
These results confirmed that JAK2V617F provides a competitive advantage to hematopoietic cells at the early stages of differentiation and, interestingly, highlighted that Trp53 inactivation associated with JAK2V617F mutation enabled an added competitive advantage to JAK2V617F mutation alone during reconstitution (stress hematopoiesis) that was not evident in BrdU experiments.

Genomic analysis reveals Trp53-dependent and -independent deregulations induced by JAK2V617F as observed in vivo
To better understand changes in immature cells related to Trp53 inactivation in a JAK2V617F context, RNA-Seq analysis was performed on steady state mice after immature populations (LT-HSC, ST-HSC, MPP, CMP, MEP, GMP) from normal, JAK2V617F/Vav-Cre, and JAK2V617F/Vav-Cre/Trp53 −/− mice (Fig. 4A) were cell sorted.
We took advantage of these data to analyze p53-related or -unrelated JAK2V617F-deregulated pathways.For p53-associated genes, a gene set enrichment analysis (GSEA) with hallmark gene sets from the molecular signature database was performed (Fig. 4D, Fig. S3B).It showed that apoptosis, TNF/NFκB signaling, and the p53 pathway were downregulated in JAK2V617FT/Trp53 −/− mice when compared with JAK2V617F mice confirming previous results illustrating the role of inflammation in TP53 inactivated cells evolution [28].This is consistent with the finding that JAK2V617F/Trp53 −/− have higher competitive engraftment.Trp53 knockout is associated with the downregulation of genes in inflammation/immune function-related pathways, including interferon response, which may be why some MPN patients harboring with P53 mutation were non-responsive to IFN treatment.
For p53-unrelated genes, we performed a GSEA analysis with ontology gene sets (M5) from the molecular signature database MsigDB (Fig. S4A).While there were few p53-unrelated genes (Table S4), there was an enrichment for cell cycle-related pathways (Fig. S4B).
These results confirm previous reports on the efficacy of IFN-α to hamper JAK2V617F/Vav-Cre cell proliferation with normalization of most hematological parameters and reduce the proliferative advantage of JAK2V617F/Vav-Cre over WT cells, but Trp53 inactivation abrogates this selective effect of IFN-α of JAK2V617F, in line with the RNA-Seq analysis.

DISCUSSION
At the biochemical level, cross-talk between JAK2V617F mutation and p53 has been largely described.JAK2V617F reduces p53 levels by inhibiting NPM1 [30].Direct interactions between p53 and STAT5 can also be inhibited by JAK2V617F [31].JAK2V617F has also been shown to induce the accumulation of La, increasing its translation and then inhibiting p53 [7].This JAK2/MDM2 pathway has been clinically reinforced since the MDM2 [9] inhibitor (Nutlin-3) significantly reduces MPN-CD34 + cell proliferation when combined with IFN-α [9].Lastly, other JAK2-dependant transduction pathways such as the RAS/MAPK or AKT/mTOR pathways also regulate p53 functions because ERK phosphorylation upregulates MDM2 expression.Taken together, these results suggest that JAK2V617F mutation and inactivation of p53 could be overlapping events in MPN, suggesting that p53 inactivation should not induce large modifications in chronic MPN phenotypes.
The fact that JAK2 directly regulates and inhibits TP53 function on the one hand but that TP53 inhibition could induce a transformation of JAK2V617F cells on the other hand prompted us to develop a more physiological model of JAK2V617F expression in the context of p53 inactivation in immature BM cells.Our analyses of the biological process and more particularly the MPN phenotype (high leukocytes, red blood cells, and platelets; spleen enlargement, BM histology, and survival) demonstrate the lack of differences between JAK2V617F/Vav-Cre and JAK2V617F/Vav-Cre/Trp53 −/− mice, at least during the first months (our severe model of MPN induces death of mice in 3 months and we can not exclude that a prolonged survival would have made possible to highlight an increased risk of transformation in JAK2V617F/Vav-Cre/Trp53 −/− mice).This finding directly indicates that the JAK2V617F-induced MPN phenotype is not affected by p53-inactivation.In this context, phenotypic modifications due to p53 inactivation in a JAK2V617F context were very mild, reinforcing the hypothesis of overlapping between JAK2V617F activation and p53 inactivation.However, in patients, TP53 mutations/inactivations are late events, and we cannot definitively rule out that the order of emergence of each mutation could impact the emergence of MPN transformation.Clonal interference could play a role in such transformations.
Second, a molecular analysis performed during a steady state tends to demonstrate that 44% (from 7% to 77% according to the studied hematopoietic subpopulations) of genes that are deregulated by JAK2V617F are affected by TP53 inactivation because immature populations of JAK2V617F/Vav-Cre/Trp53 −/− cells are closer to normal compared to JAK2V617F/Vav-Cre cells.Such a set of genes include the IFN pathway-associated genes that are clearly upregulated in the JAK2V617F context (explaining the high sensitivity of these cells to IFN therapy) and comparatively downregulated when p53 is inactivated, suggesting a lower sensitivity of these cells to IFN therapy.IFN-α treatment increases TP53 phosphorylation in cells from patients with MPN but also increases phosphorylation of STAT1, p21, PUMA, and Bak proteins, which are known to be involved in apoptotic processes [32,33].IFN-α is also known to induce apoptosis of hematopoietic stem cells by the p38 MAPK pathway activation through a p53dependent mechanism [33].This finding is consistent with our RNA-Seq analysis and in vivo data that suggest that loss of p53 in the JAK2V617F context causes reduces sensitivity of stem cells to IFN-α treatment.MDM2 inhibitor RG7112 combined with IFN-α significantly decreases the number of JAK2V617F hematopoietic colonies through the activity of the p53 pathway [8] in the mouse model, which may confirm the differential sensitivity to IFN-α therapy in JAK2V617F/Vav-Cre cells compared to WT or JAK2V617F/Vav-Cre/Trp53 −/− cells in vivo.This finding suggests that patients harboring clones with p53 mutations in chronic MPN could be less sensitive to an IFN-based regimen, at least when TP53 is inactivated.This finding will need to be confirmed in IFN-αtreated patients in order to decipher whether screening p53 mutations/inactivation should be performed before IFN-α therapy in humans, just like hypothesized by our group for the use of MDM2 modulators [34,35].
In conclusion, our results illustrate that a large number of gene expression deregulations in the JAK2V617F context are affected by p53-inactivation, but the MPN phenotype is not.This work also illustrates that P53 inactivation in the hematopoietic cells endogenous JAK2V617F context is not sufficient to directly induce the transformation of MPN despite inducing a proliferative advantage over p53 WT cells in the short-term, but we can not definitively exclude such an effect in the long-term.Our data also suggest that Trp53 inactivation could induce resistance to IFN therapy, a finding that needs to be confirmed in clinical studies.

DATA POLICY DETAILS
The datasets generated during and analysed during the current study are not publicly available due to new analysis for other purposes in our lab but the raw data are available from the corresponding author on reasonable request.

Fig. 5
Fig. 5 JAK2V617F/Vav-Cre mice respond to the interferon-α (IFN-α) treatment.A The schematic of the experiment.B-D The number of peripheral blood (PB) between IFN-α treatment and untreated mice in JAK2V617F/Vav-Cre donor mice and CD5.1 donor mice.B The number of white blood cells (WBC).C The number of platelets (PLT).D The percentage of hematocrit (HCT).Graph represents at least two independent experiments of each time point, each with 4 to 5 mice per group.E-G The percentage of CD45.2 (JAK2V617F/Vav-Cre cells) in blood chimerism of treated and untreated mice.Graph represents at least two independent experiments of each time point, each with 4 to 5 mice per group.E before treatment, F after 1 month of IFN treatment, and G after 2 months of IFN treatment.H The survival of treated and untreated mice.Data are mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6
Fig. 6 JAK2V617F/Vav-Cre Trp53 −/− mice do not respond to interferon-α (IFN-α) treatment.A The schematic of the experiment.B-D The number of peripheral blood (PB) between IFN-α-treated and untreated mice in JAK2V617F/Vav-Cre/Trp53 −/− donor mice and CD5.1 donor mice.B The number of white blood cells (WBC).C The number of platelets (PLT).D The percentage of hematocrit (HCT).Graph represents at least two independent experiments of each time point, each with 4 to 5 mice per group.E-G The percentage of CD45.2 (JAK2V617F/Vav-Cre/ Trp53 −/− cell) in blood chimerism of treated and untreated mice.Graph represents at least two independent experiments of each time point, each with 4 to 5 mice per group.E before treatment, F after 1 month of IFN treatment, and G after 2 months of IFN treatment.H The survival of treated and untreated mice.Data are mean ± SD *p < 0.05, **p < 0.01, ***p < 0.00.